A method for inducing haploids and its application in plant breeding

Abstract

The present invention relates to the field of biotechnology and plant breeding, and in particular relates to a method using plant cell proliferation regulator gene to induce haploid, and a use thereof in plant breeding. In the present invention, expression profiles of unfertilized egg cells of rice and rice embryos 5 days after fertilization are analyzed, and 2 genes are discovered to have relatively high expression, OsCPRO1 and OsCPRO2. The research shows that these two genes can be utilized to induce the production of haploid descendants. Specifically, a promoter specifically expressed in egg cells controls the gene to convert a plant, obtaining induced haploid plant material of a positive transgenic plant. Furthermore, the induced haploid plant material can be combined with a MiMe system to obtain apomixes material, and thereby generate clone seeds.

1 . A protein with haploid induction ability, among which are the POX superfamily domain and the Homeobox domain, specifically, POX, the superfamily domain containing BELL domain and SKY domain: SKYLKAAQELLDEVVSV; preferably it is a OsCPRO1 or OsCPRO2 protein, amino acid sequences are shown as in SEQ ID NO: 3 and SEQ ID NO: 6, respectively, or their orthologous genes derived from the following species, optimally greater than 90%, 95%, more than 98% or 99%: rice ( Oryza sativa ), wart-grain rice ( Oryza meyeriana  var), maize ( Zea mays ), wheat ( Triticum aestivum ), durum wheat ( Triticum turgidum  subsp), wild emmer wheat ( Triticum dicoccoides ),  urartu  wheat ( Triticum urartu ), barley ( Hordeum vulgare ), section wheat ( Aegilops tauschii ), rye ( Secale cereale ), oats ( Avena sativa ), buckwheat ( Fagopyrum esculentum ), highland barley ( Hordeum vulgare ),  semen coicis  (Coixlacryma- jobi ), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis),  sorghum  ( Sorghum bicolor ), proso millet ( Panicum miliaceum ), wild rice ( Zizania latifolia ), biotic wild rice ( Zizania palustris ),  coracana ( Eleusine coracana ), sweet potato ( Dioscorea esculenta ),  cassava  ( Manihot esculenta ), potato ( Solanum tuberosum ), yam (Dioscoreapolystachya), groundnut ( Arachis hypogaea ), cranes peanut ( Arachis duranensis ), soybean ( Glycine max ), wild soybean ( Glycine soja ), adzuki bean ( Vigna angularis ), red bean ( Vigna umbellata ), lentil bean ( Lens culinaris ), mung bean ( Vigna radiata ), cowpea ( Vigna unguiculata ), kidney bean ( Phaseolus vulgaris ), broad bean ( Vicia faba ), pea ( Pisum sativum ), jack bean ( Canavalia gladiata ), pigeonpea ( Cajanus cajan ), white lupine ( Lupinus albus ), lather bean ( Mucuna pruriens ), narrow-leafed lupine ( Lupinus angustifolius ), hairy vine bean (Calopogoniummucunoides), chickpea ( Cicer arietinum ), sesame ( Sesamum indicum ), flax (Linumusitatissimum), rapeseed ( Brassica napus ), sunflower ( Helianthus annuus ), castor bean ( Ricinus communis ), red flower ( Carthamus tinctorius ), beet ( Beta vulgaris ), sugarcane ( Saccharum officinarum ), cotton ( Gossypium  spp), marijuana ( Cannabis sativa ), jiangnan rolling cypress ( Selaginella moellendorffii ), lemon  eucalyptus  (Corymbia  citriodora ), water green tree (Tetracentronsinense), mesquite tree ( Prosopis alba ),  taxus chinensis  ( Taxus chinensis ), olive ( Olea europaea ), African  acacia  (Abrusprecatorius), switchgrass ( Panicum virgatum ), red-vein maiguo (Rhamnellarubrinervis), American hickory ( Carya illinoinensis ), tea ( Camellia sinensis ), tobacco ( Nicotiana tabacum ), elephant grass ( Pennisetum purpureum ), hard-straight ryegrass ( Lolium rigidum ), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed ( Pennisetum alopecuroides ), Sudan grass ( Sorghum sudanense ), Tang pine grass ( Thalictrum thalictroides ), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL),  Arabidopsis  ( Arabidopsis thaliana ),  Physcomitrella ( Physcomitrium patens ), hornmoss (Ceratodonpurpureus), cabbage ( Brassica oleracea ), mustard ( Brassica juncea ), Chinesegreen cabbage ( Brassica rapa ), cauliflower ( Brassica oleracea ), lettuce ( Lactuca sativa ), spinach ( Spinacia oleracea ), radish ( Raphanus sativus ), Chinesecabbage ( Brassica rapa ), zucchini ( Cucurbita pepo ), hemsley ( Apium graveolens ), leek ( Allium tuberosum ), carrot ( Daucus carota ), eggplant ( Solanum melongena ), tomato ( Lycopersicon esculentum ), cucumber ( Cucumis sativus ), wax gourd (Benincasahispida), pumpkin ( Cucurbita moschata ), loofah ( Luffa cylindrica ), vegetable melon ( Cucumis melo ), lotus vegetables ( Potentilla anserina ), day lily ( Hemerocallis citrina ), stem lettuce ( Lactuca sativa ),  asparagus  ( Asparagus officinalis ), chili pepper ( Capsicum annuum ), garlic ( Allium sativum ), spring onion ( Allium fistulosum ), Jerusalem artichoke ( Helianthus tuberosus ), Coriander ( Coriandrum sativum ), watermelon ( Citrullus lanatus ), pear ( Pyrus  spp), peach ( Prunus persica ), apricot ( Armeniaca  vulgaris), lee ( Prunus salicina ), date ( Ziziphus jujuba ), green plum (Vaticamangachapoi), apple ( Malus pumila ), sand fruit ( Malus asiatica ), cherry ( Cerasus  spp), walnut ( Juglans regia ), strawberry ( Fragaria ananassa ), grape ( Vitis vinifera ), pineapple pear ( Ananas comosus ),  coptis chinensis  ( Coptis chinensis ), Chinese milk vetch ( Astragalus sinicus ),  ginseng  ( Panax ginseng ),  Angelica sinensis  ( Angelica sinensis ), honeysuckle ( Lonicera japonica ), Ai ( Artemisia argyi ), peppermint ( Mentha canadensis ), orchid ( Cymbidium  ssp), lily ( Lilium brownii ), tulip ( Tulipa gesneriana ). 
     
     
         2 . The gene encoding a protein as described in  claim 1 , which is either OsCPRO1 or OsCPRO2, where the gene symbol of OsCPRO1 is LOC_Os03g47740, the gene symbol of OsCPRO2 is LOC_Os12g43950; Or BEL1-like homeodomain protein 6 derived from maize, Sequence ID: PWZ21223.1; Or BEL1-like homeodomain protein 7 derived from wheat, Sequence ID: XP_044365192.1; Or BEL1-like homeodomain protein 1 derived from soybean, Sequence ID: XP_003543416.1; Or BEL1-like homeodomain protein 1 from groundpeanut, Sequence ID: XP_016170518.1; Or at least 90%, 95% of the above genes, more preferably more than 98%, or 99% of the homologous genes derived from the same species, and still has the haploid induction ability. 
     
     
         3 . The gene with haploid induction ability as described in  claim 2  is characterized by the nucleotide sequence of OsCPRO1 as shown in SEQ ID NO: 1, or more than 90%, 95%, 98% and 99% of homologous genes derived from rice and still have haploid induction ability; the nucleotide sequence of OsCPRO2 as shown in SEQ ID NO: 4, preferably more than 98% and 99% of homologous genes derived from rice, and still has haploid induction ability. 
     
     
         4 . The gene having the haploid induction ability as described in  claim 2 , it is characterized by the fact that, the CDS nucleotide sequence of OsCPRO1 is as shown in SEQ ID NO: 2, or is derived from rice that still has a haploid-inducing nucleotide sequence with greater than 98% or 99% identity, and still has the haploid induction ability; the CDS nucleotide sequence of OsCPRO2 is as shown in SEQ ID NO: 5, or derived from rice still has haploid induction ability with more than 98% and 99% identity. 
     
     
         5 . An expression cassette, a recombinant vector, a recombinant cell, or a host cell containing a gene according to  claim 2 . 
     
     
         6 . The protein according to  claim 1 , wherein the plant is selected from the group consisting of rice ( Oryza sativa ), wart-grain rice ( Oryza meyeriana  var), maize ( Zea mays ), wheat ( Triticum aestivum ), durum wheat ( Triticum turgidum  subsp), wild emmer wheat ( Triticum dicoccoides ),  urartu  wheat ( Triticum urartu ), barley ( Hordeum vulgare ), section wheat ( Aegilops tauschii ), rye ( Secale cereale ), oats ( Avena sativa ), buckwheat ( Fagopyrum esculentum ), highland barley ( Hordeum vulgare ),  semen coicis  (Coixlacryma- jobi ), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis),  sorghum  ( Sorghum bicolor ), proso millet ( Panicum miliaceum ), wild rice ( Zizania latifolia ), biotic wild rice ( Zizania palustris ),  coracana  ( Eleusine coracana ), sweet potato ( Dioscorea esculenta ),  cassava  ( Manihot esculenta ), potato ( Solanum tuberosum ), yam (Dioscoreapolystachya), groundnut ( Arachis hypogaea ), cranes peanut ( Arachis duranensis ), soybean ( Glycine max ), wild soybean ( Glycine soja ), adzuki bean ( Vigna angularis ), red bean ( Vigna umbellata ), lentil bean ( Lens culinaris ), mung bean ( Vigna radiata ), cowpea ( Vigna unguiculata ), kidney bean ( Phaseolus vulgaris ), broad bean ( Vicia faba ), pea ( Pisum sativum ), jack bean ( Canavalia gladiata ), pigeonpea ( Cajanus cajan ), white lupine ( Lupinus albus ), lather bean ( Mucuna pruriens ), narrow-leafed lupine ( Lupinus angustifolius ), hairy vine bean (Calopogoniummucunoides), chickpea ( Cicer arietinum ), sesame ( Sesamum indicum ), flax (Linumusitatissimum), rapeseed ( Brassica napus ), sunflower ( Helianthus annuus ), castor bean ( Ricinus communis ), red flower ( Carthamus tinctorius ), beet ( Beta vulgaris ), sugarcane ( Saccharum officinarum ), cotton ( Gossypium  spp), marijuana ( Cannabis sativa ), jiangnan rolling cypress ( Selaginella moellendorffii ), lemon  eucalyptus  (Corymbia  citriodora ), water green tree (Tetracentronsinense), mesquite tree ( Prosopis alba ),  taxus chinensis  ( Taxus chinensis ), olive ( Olea europaea ), African  acacia (Abrusprecatorius), switchgrass ( Panicum virgatum ), red-vein maiguo (Rhamnellarubrinervis), American hickory ( Carya illinoinensis ), tea ( Camellia sinensis ), tobacco ( Nicotiana tabacum ), elephant grass ( Pennisetum purpureum ), hard-straight ryegrass ( Lolium rigidum ), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed ( Pennisetum alopecuroides ), Sudan grass ( Sorghum sudanense ), Tang pine grass ( Thalictrum thalictroides ), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL),  Arabidopsis  ( Arabidopsis thaliana ),  Physcomitrella ( Physcomitrium patens ), hornmoss (Ceratodonpurpureus), cabbage ( Brassica oleracea ), mustard ( Brassica juncea ), Chinesegreen cabbage ( Brassica rapa ), cauliflower ( Brassica oleracea ), lettuce ( Lactuca sativa ), spinach ( Spinacia oleracea ), radish ( Raphanus sativus ), Chinesecabbage ( Brassica rapa ), zucchini ( Cucurbita pepo ), hemsley ( Apium graveolens ), leek ( Allium tuberosum ), carrot ( Daucus carota ), eggplant ( Solanum melongena ), tomato ( Lycopersicon esculentum ), cucumber ( Cucumis sativus ), wax gourd (Benincasahispida), pumpkin ( Cucurbita moschata ), loofah ( Luffa cylindrica ), vegetable melon ( Cucumis melo ), lotus vegetables ( Potentilla anserina ), day lily ( Hemerocallis citrina ), stem lettuce ( Lactuca sativa ),  asparagus  ( Asparagus officinalis ), chili pepper ( Capsicum annuum ), garlic ( Allium sativum ), spring onion ( Allium fistulosum ), Jerusalem artichoke ( Helianthus tuberosus ), Coriander ( Coriandrum sativum ), watermelon ( Citrullus lanatus ), pear ( Pyrus  spp), peach ( Prunus persica ), apricot ( Armeniaca  vulgaris), lee ( Prunus salicina ), date ( Ziziphus jujuba ), green plum (Vaticamangachapoi), apple ( Malus pumila ), sand fruit ( Malus asiatica ), cherry ( Cerasus  spp), walnut ( Juglans regia ), strawberry ( Fragaria ananassa ), grape ( Vitis vinfera ), pineapple pear ( Ananas comosus ),  coptis chinensis  ( Coptis chinensis ), Chinese milk vetch ( Astragalus sinicus ),  ginseng  ( Panax ginseng ),  Angelica sinensis  ( Angelica sinensis ), honeysuckle ( Lonicera japonica ), Ai ( Artemisia argyi ), peppermint ( Mentha canadensis ), orchid ( Cymbidium  ssp), lily ( Lilium brownii ), and tulip ( Tulipa gesneriana ). 
     
     
         7 . A method of inducing plant haploids using a gene according to  claim 1 , comprising the steps of including the gene to obtain a positive transgene plant controlled by the promoter for specific expression in egg cells of the gene and, preferably, the plant including a monocot and a dicotyledon; preferred of all, the plant was selected from rice ( Oryza sativa ), wart-grain rice ( Oryza meyeriana  var), maize ( Zea mays ), wheat ( Triticum aestivum ), durum wheat ( Triticum turgidum  subsp), wild emmer wheat ( Triticum dicoccoides ),  urartu  wheat ( Triticum urartu ), barley ( Hordeum vulgare ), section wheat ( Aegilops tauschii ), rye ( Secale cereale ), oats ( Avena sativa ), buckwheat ( Fagopyrum esculentum ), highland barley ( Hordeum vulgare ),  semen coicis  (Coixlacryma- jobi ), foxtail millet (Setariaitalica), fonio millet (Digitariaexiis),  sorghum  ( Sorghum bicolor ), proso millet ( Panicum miliaceum ), wild rice ( Zizania latifolia ), biotic wild rice ( Zizania palustris ),  coracana ( Eleusine coracana ), sweet potato ( Dioscorea esculenta ),  cassava  ( Manihot esculenta ), potato ( Solanum tuberosum ), yam (Dioscoreapolystachya), groundnut ( Arachis hypogaea ), cranes peanut ( Arachis duranensis ), soybean ( Glycine max ), wild soybean ( Glycine soja ), adzuki bean ( Vigna angularis ), red bean ( Vigna umbellata ), lentil bean ( Lens culinaris ), mung bean ( Vigna radiata ), cowpea ( Vigna unguiculata ), kidney bean ( Phaseolus vulgaris ), broad bean ( Vicia faba ), pea ( Pisum sativum ), jack bean ( Canavalia gladiata ), pigeonpea ( Cajanus cajan ), white lupine ( Lupinus albus ), lather bean ( Mucuna pruriens ), narrow-leafed lupine ( Lupinus angustifolius ), hairy vine bean (Calopogoniummucunoides), chickpea ( Cicer arietinum ), sesame ( Sesamum indicum ), flax (Linumusitatissimum), rapeseed ( Brassica napus ), sunflower ( Helianthus annuus ), castor bean ( Ricinus communis ), red flower ( Carthamus tinctorius ), beet ( Beta vulgaris ), sugarcane ( Saccharum officinarum ), cotton ( Gossypium  spp), marijuana ( Cannabis sativa ), jiangnan rolling cypress ( Selaginella moellendorffii ), lemon  eucalyptus  (Corymbia  citriodora ), water green tree (Tetracentronsinense), mesquite tree ( Prosopis alba ),  taxus chinensis  ( Taxus chinensis ), olive ( Olea europaea ), African  acacia (Abrusprecatorius), switchgrass ( Panicum virgatum ), red-vein maiguo (Rhamnellarubrinervis), American hickory ( Carya illinoinensis ), tea ( Camellia sinensis ), tobacco ( Nicotiana tabacum ), elephant grass ( Pennisetum purpureum ), hard-straight ryegrass ( Lolium rigidum ), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed ( Pennisetum alopecuroides ), Sudan grass ( Sorghum sudanense ), Tang pine grass ( Thalictrum thalictroides ), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL),  Arabidopsis  ( Arabidopsis thaliana ),  Physcomitrella ( Physcomitrium patens ), hornmoss (Ceratodonpurpureus), cabbage ( Brassica oleracea ), mustard ( Brassica juncea ), Chinesegreen cabbage ( Brassica rapa ), cauliflower ( Brassica oleracea ), lettuce ( Lactuca sativa ), spinach ( Spinacia oleracea ), radish ( Raphanus sativus ), Chinesecabbage ( Brassica rapa ), zucchini ( Cucurbita pepo ), hemsley ( Apium graveolens ), leek ( Allium tuberosum ), carrot ( Daucus carota ), eggplant ( Solanum melongena ), tomato ( Lycopersicon esculentum ), cucumber ( Cucumis sativus ), wax gourd (Benincasahispida), pumpkin ( Cucurbita moschata ), loofah ( Luffa cylindrica ), vegetable melon ( Cucumis melo ), lotus vegetables ( Potentilla anserina ), day lily ( Hemerocallis citrina ), stem lettuce ( Lactuca sativa ),  asparagus  ( Asparagus officinalis ), chili pepper ( Capsicum annuum ), garlic ( Allium sativum ), spring onion (Ailium  fistulosum ), Jerusalem artichoke ( Helianthus tuberosus ), Coriander ( Coriandrum sativum ), watermelon ( Citrullus lanatus ), pear ( Pyrus  spp), peach ( Prunus persica ), apricot ( Armeniaca  vulgaris), lee ( Prunus salicina ), date ( Ziziphus jujuba ), green plum (Vaticamangachapoi), apple ( Malus pumila ), sand fruit ( Malus asiatica ), cherry ( Cerasus  spp), walnut ( Juglans regia ), strawberry ( Fragaria ananassa ), grape ( Vitis vinifera ), pineapple pear ( Ananas comosus ),  coptis chinensis  ( Coptis chinensis ), Chinese milk vetch ( Astragalus sinicus ),  ginseng  ( Panax ginseng ),  Angelica sinensis  ( Angelica sinensis ), honeysuckle ( Lonicera japonica ), Ai ( Artemisia argyi ), peppermint ( Mentha canadensis ), orchid ( Cymbidium  ssp), lily ( Lilium brownii ), tulip ( Tulipa gesneriana ). 
     
     
         8 . The method of  claim 7 , wherein the promoter for egg specific expression is a promoter of AtDD45 egg specific expression; preferably with the nucleotide sequence shown in SEQ ID NO: 7; the import is an  Agrobacterium -mediated or gene gun or gene editing method. 
     
     
         9 . The method of  claim 7 , comprising the step of taking transgenic positive material to obtain haploid material. 
     
     
         10 . The method of  claim 9 , further comprising combining haploid inductive material to obtain apomixis material with a MiMe system.